Overview: The objectives of this project are to understand the molecular biology of pain-sensing neurons and peripheral tissues at the transcriptome level and modulation of transcriptomic parameters in acute and chronic pain models and in human patients or post-mortem samples. The laboratory has established research methodology and protocols, built an infrastructure of hardware and software, formed collaborative arrangements, trained a team of scientists and support personnel to utilize the methodology of RNA-Seq, in situ hybridization and tissue procurement. We have performed hundreds of deep sequencing runs in various species and models and resulting in many billions of reads of transcriptome sequence information. We are intensively involved in the analysis of the resulting datasets from physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of inflamed peripheral tissue, axotomized dorsal root ganglion (DRG) neurons, dorsal and ventral spinal cords, peripheral nerve, and inflamed tissue. We are also investigating transcriptional processes affected by general anesthesia in higher order brain regions. Use of the newer high-throughput sequencing devices allows sampling of multiple time points to follow the evolution and resolution of the intervention with enough read depth and number of samples at each point to permit thorough assessment and statistical comparison, respectively. Because we isolated certain neuronal and non-neuronal cell populations, we know which genes are in pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, and supporting cells or Schwann cells. The ability to form incisive hypotheses regarding pain physiology is greatly advanced by this type of tissue and neuron-specific information. We now have quantitative information on all the genes that mediate DRG and spinal cord sensory and motor functions and formation of the myelin sheath which, in turn, permits us to build new levels of understanding of how pain is generated, transmitted, processed and modulated in the peripheral and central nervous systems in animal models and humans. TRPV1 Transcriptome: One important focus for our group is the subpopulation of DRG neurons that express the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans indicating a crucial role for TRPV1+ neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons, we performed deep RNA sequencing (RNA-Seq) on mouse, rat, canine, and human ganglionic preparations targeting TRPV1 neurons. We published initial reports on the comprehensive transcriptomic profile of this clinically important population of nociceptive neurons, followed by a second that distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome. In the present cycle we have extended the analysis to TRPV1+ neurons functionally identified by agonist-activated calcium fluorescence and DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX, a cohort of canines with cancer pain that were also treated for pain with RTX and controlled treatments in the rat. In combination, these data demonstrate that the most sensitive neuronal component is the centrally projecting axons that contain TRPV1 whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight was gained from transcriptomic analyses and is being used to fine-tune the administration protocol in our human clinical trial. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. The quantitative relationships between the exact genes that mediate actions of known analgesic drugs such as morphine, clonidine, lidocaine, ibuprofen, gabapentin and emerging nociceptor-neuron-specific sodium channels. This is a high resolution transformative technology that provides sequenced based counting of transcripts to create criteria for which genes are well-expressed versus those expressed at an inconsequential level. Additionally we can make qualitative assignments as to which molecular paralogs are in the nociceptive populations allowing a more informative mechanistic framework to emerge. In this cycle we identified a protein tyrosine kinase and an orphan GPCR that are well expressed and upregulated in the nociceptive population. We published a report that began with transcriptomic profiling of lipid-generating genes to define metabolic pathways for production of potentially neuroactive lipids. This novel approach predicted two new lipids which we verified using mass spectrometry. Correlative animal behavioral and human headache and psoriasis studies suggest roles in pain sensitization and itch. Transcriptomics of human pain sensitivity resulting from genetic variations: The RNA-Seq data provides a means for amplification of ongoing studies and informs all of our hypothesis-driven studies. We are in the process of characterizing human genetic copy number variations (CNVs) that produce pain insensitivity. The mechanisms can be probed, in part, using rat or mouse models of the CNVs. For example, we were able to evaluate the integrity of neurons in sensory ganglion and spinal cord using a rodent model of the hemideletion. In these studies, transcriptomic profiling allows for more precise hypothesis generation and testing. Anesthesia Transcriptome: We are in the process of completing a transcriptomic assessment of the effects of inhalation general anesthesia and ketamine infusion on cortical and hippocampal transcriptomes and associated proteins identified from the gene analysis. These are initial steps to a larger investigation of the impact of general anesthesia on cognitive function. In humans, general anesthesia can be deleterious to cognitive function. We hypothesize that mechanistic insight into the defect state can be obtained by understanding the molecular-level changes induced by anesthesia and the capacity for recovery. Our results indicate that communication between synaptic input and nuclear transcriptional control is strongly inhibited by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. We detect widespread modulation of genes that mediate functional plasticity and memory formation. Corresponding decreases in several of the proteins are also observed. The data suggest that anesthesia can transiently uncouple synaptic activity from neuronal transcriptional control. Summary: The datasets acquired over the past several years provide unprecedented and extremely fine-grained detail on gene expression in pain-sensing circuits and anesthesia-sensitive brain regions. The basic goal is to understand how we sense and control pain. We are determining exactly what molecules the different types of pain-sensing neurons make and how they work together to do their job. We have extended this approach to actions of anesthetic agents and observe a remarkable sensitivity of cerebral cortex to gaseous anesthetics compared to hippocampus. This suggests that executive function and working memory will be the most susceptible variables affected by general anesthesia. Taken together our data provide a transformative new resource for the pain research and anesthesia communities, and will allow more precise assessment and verification of experimental and clinical results.